Elongation factor P (EF-P) plays a critical role in protein synthesis in bacteria. Loss of EF- P leads to a variety of phenotypes, including metabolic stress, altered antibiotic resistance, and loss of virulence. Although EF-P was discovered in the 1970's, the first compelling explanation of its biological significance appeared in early 2013. Protein synthesis pauses when three or more consecutive prolines occur in the nascent peptide; EF-P binds to the ribosome and restores rapid peptide synthesis. Our goal is to achieve a molecular-level understanding of how polyproline motifs pause translation and how pauses are resolved by EF-P. These studies will shed light on how the nascent peptide modulates translational rates, a phenomenon with important implications for protein folding and gene regulation. The molecular mechanism of pausing is poorly understood. We hypothesize that Pro alters the geometry of key nucleotides in the ribosomal active site, inhibiting catalysis. In Aim 1 we will test this hypothesis using pre- steady state kinetic methods to isolate rate defects that lead to pausing. We will probe the interaction of the peptide and active site using amino acid analogs and rRNA mutations. We will also investigate how EF-P alleviates ribosome pausing. EF-P has a tRNA-like structure and interacts with peptidyl-tRNA within the ribosome. The Lys34 side chain, positioned near the peptidyl-transferase center, is covalently modified with a -lysyl moiety that is essential for EF- P function in E. coli. We hypothesize that the modified side chain subtly rearranges the geometry of the ribosomal active site to restore catalysis. In Aim 2, we will test the role of the -lysyl moiety by chemically modifying EF-P with a series of -lysine analogs. Notably, the enzymes responsible for -lysylation, YjeA and YjeK, are lacking in 70% of bacteria. We will determine how EF-P is modified in two of these species and identify the enzymes responsible. Finally, our biochemical data suggest that EF-P alleviates translational pausing at several motifs, not just polyproline stretches. In Aim 3, we will define the scope of EF-P activity using ribosome profiling, a method for monitoring translation globally in living cell. By monitoring changes in translation and mRNA expression, we will link pausing to the pleiotropic phenotypes observed in cells lacking EF-P.